The hyphal-specific toxin candidalysin promotes fungal gut commensalism.

The fungus Candida albicans frequently colonizes the human gastrointestinal tract, from which it can disseminate to cause systemic disease. This polymorphic species can transition between growing as single-celled yeast and as multicellular hyphae to adapt to its environment. The current dogma of C. albicans commensalism is that the yeast form is optimal for gut colonization, whereas hyphal cells are detrimental to colonization but critical for virulence1-3. Here, we reveal that this paradigm does not apply to multi-kingdom communities in which a complex interplay between fungal morphology and bacteria dictates C. albicans fitness. Thus, whereas yeast-locked cells outcompete wild-type cells when gut bacteria are absent or depleted by antibiotics, hyphae-competent wild-type cells outcompete yeast-locked cells in hosts with replete bacterial populations. This increased fitness of wild-type cells involves the production of hyphal-specific factors including the toxin candidalysin4,5, which promotes the establishment of colonization. At later time points, adaptive immunity is engaged, and intestinal immunoglobulin A preferentially selects against hyphal cells1,6. Hyphal morphotypes are thus under both positive and negative selective pressures in the gut. Our study further shows that candidalysin has a direct inhibitory effect on bacterial species, including limiting their metabolic output. We therefore propose that C. albicans has evolved hyphal-specific factors, including candidalysin, to better compete with bacterial species in the intestinal niche.

Filamentation and biofilm formation are regulated by the phase-separation capacity of network transcription factors in Candida albicans.

The ability of the fungus Candida albicans to filament and form biofilms contributes to its burden as a leading cause of hospital-acquired infections. Biofilm development involves an interconnected transcriptional regulatory network (TRN) consisting of nine transcription factors (TFs) that bind both to their own regulatory regions and to those of the other network TFs. Here, we show that seven of the nine TFs in the C. albicans biofilm network contain prion-like domains (PrLDs) that have been linked to the ability to form phase-separated condensates. Construction of PrLD mutants in four biofilm TFs reveals that these domains are essential for filamentation and biofilm formation in C. albicans. Moreover, biofilm PrLDs promote the formation of phase-separated condensates in the nuclei of live cells, and PrLD mutations that abolish phase separation (such as the removal of aromatic residues) also prevent biofilm formation. Biofilm TF condensates can selectively recruit other TFs through PrLD-PrLD interactions and can co-recruit RNA polymerase II, implicating condensate formation in the assembly of active transcriptional complexes. Finally, we show that PrLD mutations that block the phase separation of biofilm TFs also prevent filamentation in an in vivo model of gastrointestinal colonization. Together, these studies associate transcriptional condensates with the regulation of filamentation and biofilm formation in C. albicans, and highlight how targeting of PrLD-PrLD interactions could prevent pathogenesis by this species.

The Candida albicans reference strain SC5314 contains a rare, dominant allele of the transcription factor Rob1 that modulates filamentation, biofilm formation, and oral commensalism.

IMPORTANCE: Candida albicans is a commensal fungus that colonizes the human oral cavity and gastrointestinal tract but also causes mucosal as well as invasive disease. The expression of virulence traits in C. albicans clinical isolates is heterogeneous and the genetic basis of this heterogeneity is of high interest. The C. albicans reference strain SC5314 is highly invasive and expresses robust filamentation and biofilm formation relative to many other clinical isolates. Here, we show that SC5314 derivatives are heterozygous for the transcription factor Rob1 and contain an allele with a rare gain-of-function SNP that drives filamentation, biofilm formation, and virulence in a model of oropharyngeal candidiasis. These findings explain, in part, the outlier phenotype of the reference strain and highlight the role heterozygosity plays in the strain-to-strain variation of diploid fungal pathogens.

The Candida albicans reference strain SC5314 contains a rare, dominant allele of the transcription factor Rob1 that modulates biofilm formation and oral commensalism.

Candida albicans is a diploid human fungal pathogen that displays significant genomic and phenotypic heterogeneity over a range of virulence traits and in the context of a variety of environmental niches. Here, we show that the effects of Rob1 on biofilm and filamentation virulence traits is dependent on both the specific environmental condition and the clinical strain of C. albicans . The C. albicans reference strain SC5314 is a ROB1 heterozygote with two alleles that differ by a single nucleotide polymorphism at position 946 resulting in a serine or proline containing isoform. An analysis of 224 sequenced C. albicans genomes indicates that SC5314 is the only ROB1 heterozygote documented to date and that the dominant allele contains a proline at position 946. Remarkably, the ROB1 alleles are functionally distinct and the rare ROB1 946S allele supports increased filamentation in vitro and increased biofilm formation in vitro and in vivo, suggesting it is a phenotypic gain-of-function allele. SC5314 is amongst the most highly filamentous and invasive strains characterized to date. Introduction of the ROB1 946S allele into a poorly filamenting clinical isolate increases filamentation and conversion of an SC5314 laboratory strain to a ROB1 946S homozygote increases in vitro filamentation and biofilm formation. In a mouse model of oropharyngeal infection, the predominant ROB1 946P allele establishes a commensal state while the ROB1 946S phenocopies the parent strain and invades into the mucosae. These observations provide an explanation for the distinct phenotypes of SC5314 and highlight the role of heterozygosity as a driver of C. albicans phenotypic heterogeneity. Importance: Candida albicans is a commensal fungus that colonizes human oral cavity and gastrointestinal tracts but also causes mucosal as well as invasive disease. The expression of virulence traits in C. albicans clinical isolates is heterogenous and the genetic basis of this heterogeneity is of high interest. The C. albicans reference strain SC5314 is highly invasive and expresses robust filamentation and biofilm formation relative to many other clinical isolates. Here, we show that SC5314 derivatives are heterozygous for the transcription factor Rob1 and contain an allele with a rare gain-of-function SNP that drives filamentation, biofilm formation, and virulence in a model of oropharyngeal candidiasis. These finding explain, in part, the outlier phenotype of the reference strain and highlight the role of heterozygosity plays in the strain-to-strain variation of diploid fungal pathogens.

Rewilding of laboratory mice enhances granulopoiesis and immunity through intestinal fungal colonization.

The paucity of blood granulocyte populations such as neutrophils in laboratory mice is a notable difference between this model organism and humans, but the cause of this species-specific difference is unclear. We previously demonstrated that laboratory mice released into a seminatural environment, referred to as rewilding, display an increase in blood granulocytes that is associated with expansion of fungi in the gut microbiota. Here, we find that tonic signals from fungal colonization induce sustained granulopoiesis through a mechanism distinct from emergency granulopoiesis, leading to a prolonged expansion of circulating neutrophils that promotes immunity. Fungal colonization after either rewilding or oral inoculation of laboratory mice with Candida albicans induced persistent expansion of myeloid progenitors in the bone marrow. This increase in granulopoiesis conferred greater long-term protection from bloodstream infection by gram-positive bacteria than by the trained immune response evoked by transient exposure to the fungal cell wall component ß-glucan. Consequently, introducing fungi into laboratory mice may restore aspects of leukocyte development and provide a better model for humans and free-living mammals that are constantly exposed to environmental fungi.

Glucose depletion enables Candida albicans mating independently of the epigenetic white-opaque switch.

The human fungal pathogen Candida albicans can switch stochastically and heritably between a "white" phase and an "opaque" phase. Opaque cells are the mating-competent form of the species, whereas white cells are thought to be essentially "sterile". Here, we report that glucose depletion, a common nutrient stress, enables C. albicans white cells to undergo efficient sexual mating. The relative expression levels of pheromone-sensing and mating-associated genes (including STE2/3, MFA1, MFα1, FIG1, FUS1, and CEK1/2) are increased under glucose depletion conditions, while expression of mating repressors TEC1 and DIG1 is decreased. Cph1 and Tec1, factors that act downstream of the pheromone MAPK pathway, play opposite roles in regulating white cell mating as TEC1 deletion or CPH1 overexpression promotes white cell mating. Moreover, inactivation of the Cph1 repressor Dig1 increases white cell mating ~4000 fold in glucose-depleted medium relative to that in the presence of glucose. Our findings reveal that the white-to-opaque epigenetic switch may not be a prerequisite for sexual mating in C. albicans in nature.

Aneuploidy and gene dosage regulate filamentation and host colonization by Candida albicans.

Aneuploidy is a frequent occurrence in fungal species where it can alter gene expression and promote adaptation to a variety of environmental cues. Multiple forms of aneuploidy have been observed in the opportunistic fungal pathogen Candida albicans, which is a common component of the human gut mycobiome but can escape this niche and cause life-threatening systemic disease. Using a barcode sequencing (Bar-seq) approach, we evaluated a set of diploid C. albicans strains and found that a strain carrying a third copy of chromosome (Chr) 7 was associated with increased fitness during both gastrointestinal (GI) colonization and systemic infection. Our analysis revealed that the presence of a Chr 7 trisomy resulted in decreased filamentation, both in vitro and during GI colonization, relative to isogenic euploid controls. A target gene approach demonstrated that NRG1, encoding a negative regulator of filamentation located on Chr 7, contributes to increased fitness of the aneuploid strain due to inhibition of filamentation in a gene dosage-dependent fashion. Together, these experiments establish how aneuploidy enables the reversible adaptation of C. albicans to its host via gene dosage-dependent regulation of morphology.

Phase separation in fungi.

Phase separation, in which macromolecules partition into a concentrated phase that is immiscible with a dilute phase, is involved with fundamental cellular processes across the tree of life. We review the principles of phase separation and highlight how it impacts diverse processes in the fungal kingdom. These include the regulation of autophagy, cell signalling pathways, transcriptional circuits and the establishment of asymmetry in fungal cells. We describe examples of stable, phase-separated assemblies including membraneless organelles such as the nucleolus as well as transient condensates that also arise through phase separation and enable cells to rapidly and reversibly respond to important environmental cues. We showcase how research into phase separation in model yeasts, such as Saccharomyces cerevisiae and Schizosaccharomyces pombe, in conjunction with that in plant and human fungal pathogens, such as Ashbya gossypii and Candida albicans, is continuing to enrich our understanding of fundamental molecular processes.

Friendly fungi: symbiosis with commensal Candida albicans.

Mucosal tissues are constitutively colonized by a wide assortment of host-adapted microbes. This includes the polymorphic fungus Candida albicans which is a primary target of human adaptive responses. Immunogenicity is replicated after intestinal colonization in preclinical models with a surprising array of protective benefits for most hosts, but harmful consequences for a few. The interaction between fungus and host is complex, and traditionally, the masking of antigenic fungal ligands has been viewed as a tactic for fungal immune evasion during invasive infection. However, we propose that dynamic expression of cell wall moieties, host cell lysins, and other antigenic C. albicans determinants is necessary during the more ubiquitous context of intestinal colonization to prime immunogenicity and optimize mammalian host symbiosis.

An Adjuvant-Based Approach Enables the Use of Dominant HYG and KAN Selectable Markers in Candida albicans.

Candida albicans is a pathobiont fungus that can colonize multiple niches in the human body but is also a frequent cause of both mucosal and systemic disease. Despite its clinical importance, a paucity of dominant selectable markers has hindered the development of tools for genetic manipulation of the species. One factor limiting the utilization of dominant selectable markers is that C. albicans is inherently more resistant to antibiotics used for selection in other species. Here, we showed that the inclusion of suitable adjuvants can enable the use of two aminoglycoside antibiotics, hygromycin B and G418, for positive selection in C. albicans. Combining these antibiotics with an adjuvant, such as quinine or molybdate, substantially suppressed the background growth of C. albicans, thereby enabling transformants expressing CaHygB or CaKan markers to be readily identified. We verified that these adjuvants were not mutagenic to C. albicans and that CaHygB and CaKan markers were orthogonal to the existing marker NAT1/SAT1, and so provide complementary tools for the genetic manipulation of C. albicans strains. Our study also established that adjuvant-based approaches can enable the use of selectable markers that would otherwise be limited by high background growth from susceptible cells. IMPORTANCE Only a single dominant selectable marker has been widely adopted for use in the opportunistic fungal pathogen Candida albicans. This is in stark contrast to model fungi where a repertoire of dominant markers is readily available. A limiting factor for C. albicans has been the high levels of background growth obtained with multiple antibiotics, thereby limiting their use for distinguishing cells that carry an antibiotic-resistance gene from those that do not. Here, we demonstrated that the inclusion of adjuvants can reduce background growth and enable the robust use of both CaHygB and CaKan markers for genetic selection in C. albicans.

Candida albicans oscillating UME6 expression during intestinal colonization primes systemic Th17 protective immunity.

Systemic immunity is stringently regulated by commensal intestinal microbes, including the pathobiont Candida albicans. This fungus utilizes various transcriptional and morphological programs for host adaptation, but how this heterogeneity affects immunogenicity remains uncertain. We show that UME6, a transcriptional regulator of filamentation, is essential for intestinal C. albicans-primed systemic Th17 immunity. UME6 deletion and constitutive overexpression strains are non-immunogenic during commensal colonization, whereas immunogenicity is restored by C. albicans undergoing oscillating UME6 expression linked with ß-glucan and mannan production. In turn, intestinal reconstitution with these fungal cell wall components restores protective Th17 immunity to mice colonized with UME6-locked variants. These fungal cell wall ligands and commensal C. albicans stimulate Th17 immunity through multiple host pattern recognition receptors, including Toll-like receptor 2 (TLR2), TLR4, Dectin-1, and Dectin-2, which work synergistically for colonization-induced protection. Thus, dynamic gene expression fluctuations by C. albicans during symbiotic colonization are essential for priming host immunity against disseminated infection.

Candida albicans Isolates 529L and CHN1 Exhibit Stable Colonization of the Murine Gastrointestinal Tract.

Candida albicans is a pathobiont that colonizes multiple niches in the body including the gastrointestinal (GI) tract but is also responsible for both mucosal and systemic infections. Despite its prevalence as a human commensal, the murine GI tract is generally refractory to colonization with the C. albicans reference isolate SC5314. Here, we identify two C. albicans isolates, 529L and CHN1, that stably colonize the murine GI tract in three different animal facilities under conditions where SC5314 is lost from this niche. Analysis of the bacterial microbiota did not show notable differences among mice colonized with the three C. albicans strains. We compared the genotypes and phenotypes of these three strains and identified thousands of single nucleotide polymorphisms (SNPs) and multiple phenotypic differences, including their ability to grow and filament in response to nutritional cues. Despite striking filamentation differences under laboratory conditions, however, analysis of cell morphology in the GI tract revealed that the three isolates exhibited similar filamentation properties in this in vivo niche. Notably, we found that SC5314 is more sensitive to the antimicrobial peptide CRAMP, and the use of CRAMP-deficient mice modestly increased the ability of SC5314 to colonize the GI tract relative to CHN1 and 529L. These studies provide new insights into how strain-specific differences impact C. albicans traits in the host and advance CHN1 and 529L as relevant strains to study C. albicans pathobiology in its natural host niche. IMPORTANCE Understanding how fungi colonize the GI tract is increasingly recognized as highly relevant to human health. The animal models used to study Candida albicans commensalism commonly rely on altering the host microbiome (via antibiotic treatment or defined diets) to establish successful GI colonization by the C. albicans reference isolate SC5314. Here, we characterize two C. albicans isolates that can colonize the murine GI tract without antibiotic treatment and can therefore be used as tools for studying fungal commensalism. Importantly, experiments were replicated in three different animal facilities and utilized three different mouse strains. Differential colonization between fungal isolates was not associated with alterations in the bacterial microbiome but rather with distinct responses to CRAMP, a host antimicrobial peptide. This work emphasizes the importance of C. albicans intraspecies variation as well as host antimicrobial defense mechanisms in defining the outcome of commensal interactions.

Adaptation to the dietary sugar D-tagatose via genome instability in polyploid Candida albicans cells.

The opportunistic fungal pathogen Candida albicans undergoes an unusual parasexual cycle wherein diploid cells mate to form tetraploid cells that can generate genetically diverse progeny via a nonmeiotic program of chromosome loss. The genetic diversity afforded by parasex impacts clinically relevant features including drug resistance and virulence, and yet the factors influencing genome instability in C. albicans are not well defined. To understand how environmental cues impact genome instability, we monitored ploidy change following tetraploid cell growth in a panel of different carbon sources. We found that growth in one carbon source, D-tagatose, led to high levels of genomic instability and chromosome loss in tetraploid cells. This sugar is a stereoisomer of L-sorbose which was previously shown to promote karyotypic changes in C. albicans. However, while expression of the SOU1 gene enabled utilization of L-sorbose, overexpression of this gene did not promote growth in D-tagatose, indicating differences in assimilation of the two sugars. In addition, genome sequencing of multiple progenies recovered from D-tagatose cultures revealed increased relative copy numbers of chromosome 4, suggestive of chromosome-level regulation of D-tagatose metabolism. Together, these studies identify a novel environmental cue that induces genome instability in C. albicans, and further implicate chromosomal changes in supporting metabolic adaptation in this species.

Intraspecies Transcriptional Profiling Reveals Key Regulators of Candida albicans Pathogenic Traits.

The human commensal and opportunistic fungal pathogen Candida albicans displays extensive genetic and phenotypic variation across clinical isolates. Here, we performed RNA sequencing on 21 well-characterized isolates to examine how genetic variation contributes to gene expression differences and to link these differences to phenotypic traits. C. albicans adapts primarily through clonal evolution, and yet hierarchical clustering of gene expression profiles in this set of isolates did not reproduce their phylogenetic relationship. Strikingly, strain-specific gene expression was prevalent in some strain backgrounds. Association of gene expression with phenotypic data by differential analysis, linear correlation, and assembly of gene networks connected both previously characterized and novel genes with 23 C. albicans traits. Construction of de novo gene modules produced a gene atlas incorporating 67% of C. albicans genes and revealed correlations between expression modules and important phenotypes such as systemic virulence. Furthermore, targeted investigation of two modules that have novel roles in growth and filamentation supported our bioinformatic predictions. Together, these studies reveal widespread transcriptional variation across C. albicans isolates and identify genetic and epigenetic links to phenotypic variation based on coexpression network analysis.IMPORTANCE Infectious fungal species are often treated uniformly despite clear evidence of genotypic and phenotypic heterogeneity being widespread across strains. Identifying the genetic basis for this phenotypic diversity is extremely challenging because of the tens or hundreds of thousands of variants that may distinguish two strains. Here, we use transcriptional profiling to determine differences in gene expression that can be linked to phenotypic variation among a set of 21 Candida albicans isolates. Analysis of this transcriptional data set uncovered clear trends in gene expression characteristics for this species and new genes and pathways that were associated with variation in pathogenic processes. Direct investigation confirmed functional predictions for a number of new regulators associated with growth and filamentation, demonstrating the utility of these approaches in linking genes to important phenotypes.

Comparative genomics of white and opaque cell states supports an epigenetic mechanism of phenotypic switching in Candida albicans.

Several Candida species can undergo a heritable and reversible transition from a 'white' state to a mating proficient 'opaque' state. This ability relies on highly interconnected transcriptional networks that control cell-type-specific gene expression programs over multiple generations. Candida albicans, the most prominent pathogenic Candida species, provides a well-studied paradigm for the white-opaque transition. In this species, a network of at least eight transcriptional regulators controls the balance between white and opaque states that have distinct morphologies, transcriptional profiles, and physiological properties. Given the reversible nature and the high frequency of white-opaque transitions, it is widely assumed that this switch is governed by epigenetic mechanisms that occur independently of any changes in DNA sequence. However, a direct genomic comparison between white and opaque cells has yet to be performed. Here, we present a whole-genome comparative analysis of C. albicans white and opaque cells. This analysis revealed rare genetic changes between cell states, none of which are linked to white-opaque switching. This result is consistent with epigenetic mechanisms controlling cell state differentiation in C. albicans and provides direct evidence against a role for genetic variation in mediating the switch.

Epigenetic cell fate in Candida albicans is controlled by transcription factor condensates acting at super-enhancer-like elements.

Cell identity in eukaryotes is controlled by transcriptional regulatory networks that define cell-type-specific gene expression. In the opportunistic fungal pathogen Candida albicans, transcriptional regulatory networks regulate epigenetic switching between two alternative cell states, 'white' and 'opaque', that exhibit distinct host interactions. In the present study, we reveal that the transcription factors (TFs) regulating cell identity contain prion-like domains (PrLDs) that enable liquid-liquid demixing and the formation of phase-separated condensates. Multiple white-opaque TFs can co-assemble into complex condensates as observed on single DNA molecules. Moreover, heterotypic interactions between PrLDs support the assembly of multifactorial condensates at a synthetic locus within live eukaryotic cells. Mutation of the Wor1 TF revealed that substitution of acidic residues in the PrLD blocked its ability to phase separate and co-recruit other TFs in live cells, as well as its function in C. albicans cell fate determination. Together, these studies reveal that PrLDs support the assembly of TF complexes that control fungal cell identity and highlight parallels with the 'super-enhancers' that regulate mammalian cell fate.

Corrigendum: Genetic Modification of Closely Related Candida Species.

[This corrects the article DOI: 10.3389/fmicb.2019.00357.].

Horner's Syndrome with Unilateral Brachial Plexus Blockade Mimicking Cerebrovascular Accident Following Lumbar Combined Spinal Epidural Analgesia for Labor.

Horner's Syndrome (HS) with brachial plexus involvement following lumbar epidural anesthesia may be a startling event for the patient and providers. We present a case of Horner's Syndrome and complete brachial plexus blockade following epidural bolus of lidocaine for labor analgesia that was initially misdiagnosed as a cerebrovascular accident. Use of the catheter was discontinued and the episode resolved with expectant management. This complication is otherwise benign and resolves without sequelae.